PB2475: SECRETED FACTORS FROM BONE MARROW-DERIVED FIBROBLASTS PROTECT AML CELLS AGAINST APOPTOSIS MEDIATED BY A NOVEL CDK/MCL-1 INHIBITOR VIA STAT3/MCL-1 SIGNALLING
Authors: Katerina Miari, Monica Guzman, Helen Wheadon, Mark Williams
Journal: HemaSphere (2023)

Topic: 23. Hematopoiesis, stem cells and microenvironment Background: Within the bone marrow (BM), acute myeloid leukaemia (AML) blasts exist alongside BM-resident cells (e.g., fibroblasts) and via cellular crosstalk create an AML-biased microenvironment that supports AML blasts at the expense of normal haematopoiesis. Importantly, BM fibroblasts have been shown to protect leukaemic cells against the killing effects of therapy via direct fibroblast-to-AML cell contact and the induction of pro-survival signalling. While the role of direct fibroblast-to-AML cell contact in mediating fibroblast-driven protection in leukaemic cells is well established, less is known regarding the role of fibroblast secreted factors (SFs). Aims: Our aim was to investigate the ability of fibroblast SFs to protect AML cell lines against the killing effects of a novel therapeutic compound. This compound is a direct multi-cyclin-dependent kinase inhibitor/indirect MCL-1 inhibitor (CDKi/MCL-1i). The IL-6/JAK/STAT3 signalling axis was targeted as a potential method to combat fibroblast-driven resistance to CDKi/MCL-1i. Methods: The BM-derived HS-5 fibroblast cell line was cultured for 72 hours to generate conditioned media (CM), enriched in HS-5 SFs. The U937 AML cell line was exposed to cell-free HS-5 CM or recombinant human IL-6 (rhIL-6), in the presence of CDKi/MCL-1i (1μM) and apoptosis was assessed by eFluor 450/Annexin V staining and flow cytometry. Activation of STAT3 and MCL-1 in U937 cells in the presence of increasing concentrations of HS-5 CM was assessed by immunoblotting of whole cell lysates. The anti-IL-6 receptor alpha monoclonal antibody tocilizumab and the selective STAT3 degrader SD-36 were used to assess the re-sensitisation of U937 cells to the killing effects of CDKi/MCL-1i during culture with HS-5 CM. Finally, a dynamic Fluidigm array was performed to evaluate alterations in the expression of genes involved in cell cycle progression, regulation of apoptosis and various signalling axes in U937 cells cultured with HS-5 CM in the presence of CDKi/MCL-1i, SD-36 or the combination treatment. Results: At 48 hours of culture, HS-5 CM protected U937 cells against CDKi/MCL-1i-induced apoptosis across all concentrations of CM assessed (1%-50%). The protection correlated with the dose-dependent induction of phosphorylated STAT3 and its downstream target MCL-1, an anti-apoptotic protein. An ELISA and a human cytokine array confirmed the secretion of IL-6 from HS-5 cells, and IL-6 is known to induce JAK/STAT3 signalling. Indeed, addition of rhIL-6 during U937 culture partially abrogated the killing effects of CDKi/MCL-1i, suggesting that IL-6 present in HS-5 CM was responsible for the observed protection against this compound. However, blockade of the IL-6 signalling axis at the receptor level (tocilizumab) and downstream of the IL-6 receptor (SD-36) failed to re-sensitise leukaemic cells to CDKi/MCL-1i-induced apoptosis in the presence of HS-5 CM. Expression of both STAT3 and MCL-1 was downregulated by CDKi/MCL-1i, SD-36 or the combination thereof, suggesting that an alternative signalling pathway(s) is/are driving resistance to these compounds. Summary/Conclusion: Therapeutic targeting of the IL-6 signalling axis failed to overcome fibroblast SF-driven resistance to CDKi/MCL-1i in U937 cells. These findings are in line with data from the human cytokine array, demonstrating the release of multiple cytokines and growth factors from HS-5 cells. We propose that multiple signalling axes are altered in response to HS-5 CM and they most likely synergise to promote therapy resistance in AML. Keywords: Mcl-1, Acute myeloid leukemia, Bone marrow microenvironment, Drug resistance